Biotechnology Advances 27 (2009) 744–752

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Research review paper Improving salinity tolerance of through conventional breeding and genetic engineering: An analytical comparison

Muhammad Ashraf ⁎, Nudrat Aisha Akram

Department of Botany, University of Agriculture, Faiasalabad 38040, Pakistan article info abstract

Article history: The last century has witnessed a substantial improvement in yield potential, quality and disease resistance in Received 18 February 2009 crops. This was indeed the outcome of conventional breeding, which was achieved with little or no Received in revised form 26 May 2009 knowledge of underlying physiological and biochemical phenomena related to a trait. Also the resources Accepted 26 May 2009 utilized on programs involving conventional breeding were not of great magnitude. breeders have also Available online 10 June 2009 been successful during the last century in producing a few salt-tolerant cultivars/lines of some potential crops through conventional breeding, but this again has utilized modest resources. However, this approach Keywords: fi Genetic engineering seems now inef cient due to a number of reasons, and alternatively, genetic engineering for improving crop Salt tolerance salt tolerance is being actively followed these days by the plant scientists, world-over. A large number of Conventional breeding transgenic lines with enhanced salt tolerance of different crops can be deciphered from the literature but up Ion homeostasis to now only a very few field-tested cultivars/lines are known despite the fact that considerable resources Osmoprotectants have been expended on the sophisticated protocols employed for generating such transgenics. This review Antioxidants analytically compares the achievements made so far in terms of producing salt-tolerant lines/cultivars through conventional breeding or genetic engineering. © 2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 744 2. Selection and breeding for salinity tolerance — A conventional approach ...... 745 3. Engineering plants for enhanced salt tolerance: transgenic approach...... 745 4. Transgenic plants for ion transporters ...... 745 5. Transgenic plants for compatible organic solutes ...... 747 6. Transgenic plants for enhanced antioxidant production ...... 747 7. Challenges and prospects ...... 750 References ...... 751

1. Introduction “biotic approach” (Epstein, 1977; Kingsbury and Epstein, 1984; Ashraf, 1994). The term “biosaline agriculture” is also analogous to this. The Although salinity is a major problem for agriculture in arid and biotic approach was considered as the most efficient and economical semi-arid regions of the world, this menace can be observed in other means of utilizing salt-affected soils by many scientists (Epstein, 1977; areas as well. This environmental adversary has caused considerable Shannon, 1985; Ashraf, 1994) and hence a permanent solution of the crop yield losses and hence very low crop productivity. However, to problem. combat this peril, long ago, two key strategies for the utilization of Producing crops tolerant to salinity stress is imperative to run into salt-affected lands, i.e., reclamation of salt-affected soils and growing the growing food demand through sustainable agriculture. This could be halophytes or salt-tolerant crops/cultivars on such soils, were achieved either through conventional selection and breeding or through proposed (Epstein, 1977). The latter approach was referred to as the modern molecular biology approaches. Plant breeders have succeeded to some extent in producing salt-tolerant lines/cultivars of some crops through conventional breeding, but the main reason of the limited ⁎ Corresponding author. Tel.: +92 419200312. success through this strategy has been due to the fact that the magnitude E-mail address: [email protected] (M. Ashraf). of genetic variation in the gene pools of most important crops is very

0734-9750/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2009.05.026 M. Ashraf, N.A. Akram / Biotechnology Advances 27 (2009) 744–752 745 low. However, alternatively, transgenic approach for improving crop salt are special in a way that they are developed from only a single plant tolerance is being actively pursued these days by the plant scientists, cell. Transgenic plants have been developed through different genetic world-over, and a number of transgenic lines of different crops can be engineering techniques to contain desirable traits, including resis- easily deciphered from the literature. tance to pests, pesticides, diseases or adverse environmental condi- This review analytically compares the achievements made so far in tions, improved nutritional value, and enhanced product shelflife. terms of producing salt-tolerant lines/cultivars through conventional Despite a number of social, political and legal concerns, many breeding or genetic engineering. It also highlights some of the countries are now allowing transgenic crop production in conjunction challenges confronting genetic engineers because one can easily with their conventional crop production. In view of a report by The assess that only few lines/cultivars of different crops generated International Service for the Acquisition of Agro-Biotech Applications through genetic engineering and molecular biology approaches have (ISAAA) the top five countries growing transgenic crops in 2005, were thrived well under natural field conditions, though most of the claims USA, Argentina, Brazil, Canada, and China (ISAAA, 2006). However, of the scientists regarding the good performance of salt-tolerant transgenic approach is being effectively pursued by plant scientists transgenic lines are based on growth room and glasshouse trials. these days not only to improve quality and yield but also to increase tolerance to biotic and abiotic stresses in a number of crops. Since the 2. Selection and breeding for salinity tolerance — A salt tolerance trait involves a number of minor genes (quantitative conventional approach trait loci) so it is very complex. Thus, improving crop salt tolerance by genetic engineering is not so easy. Nonetheless, for improving salt Although considerable progress was made during the 20th century tolerance trait in various crops through genetic engineering plant to improve crop yield and quality through conventional breeding, we biologists have focused much on genes that encode: ion transport can see relatively little work on improving the resistance of crops proteins, compatible organic solutes, antioxidants, heat-shock and against abiotic stresses, especially salinity stress. Perhaps this has been late embryogenesis abundant proteins, and transcription factors for due to the main reason that plant breeders focused more on improving gene regulation (Ashraf et al., 2008). However, in the present review crop yield and quality than on improving stress tolerance. Nonetheless, we have discussed only the transgenic lines produced for the plant breeders had been carrying out various breeding programs overexpression of ion transport proteins, compatible organic solutes, during the last century wherein they had wisely exploited the genetic and antioxidants. variation of crops at intra-specific, inter-specific, and inter-generic levels so as to produce salt-tolerant lines/cultivars. Resultantly, they 4. Transgenic plants for ion transporters were able to release a few salt-tolerant lines/cultivars of different potential crops through conventional breeding. It is imperative to note High levels of salt in the growth medium of plants affect their that most of the lines/cultivars produced through conventional growth and development and ultimately, the yield. To prevent salt breeding were tested under natural field conditions. For example, damage, plants have developed different mechanisms to reduce Na+ some lines/cultivars of alfalfa (Medicago sativa L.) such as AZ-Germ Salt uptake or compartmentalize Na+ into vacuoles. In view of a number of II (Dobrenz et al., 1989), AZ-90NDC-ST (Johnson et al., 1991), AZ- reports it is evident that Na+ moves passively through a general cation 97MEC and AZ-97MEC-ST (Al-Doss and Smith, 1998), ZS-9491 and ZS- channel from the saline growth medium into the cytoplasm of plant 9592 (Dobrenz, 1999) were tested under natural field conditions. cells (Blumwald, 2000; Jacoby,1999; Mansour et al., 2003; Ashraf et al., Similarly, two salt-tolerant lines/cultivars of bread (Triticum 2008), but there is also a sound evidence that Na+ can move actively aestivum L.) such as S24 (Ashraf and O'Leary, 1996) and KRL1-4 through Na+/H+ antiporters, which exchange Na+ or Li+ for H+ (Hollington, 2000) were evaluated on natural salt-affected soils. (Ratner and Jacoby, 1976; Niu et al., 1993; Shi et al., 2003), and the These few examples depict that there has been a limited success in regulation of Na+ uptake and accumulation into plant cells is relied on producing salt-tolerant cultivars of different potential crops using the the regulation of proton pumps and antiporters operating at both conventional breeding approach. The main problem conventional plant plasma membrane and tonoplast (Ashraf, 2004). Na+/H+ antiporters breeders have been facing is the low magnitude of genetically based are found in every biological kingdom, from bacteria to humans to variation in the gene pools of most crop . Thus, one cannot expect higher plants (Padan et al., 2001). There are several families of Na+/H+ substantial improvements in crop salt tolerance. Under such circum- antiporters including NhaA, NhaB, NhaC, NhaD, and NapA in stances, it is advisable to utilize salt-tolerant wild relatives of crop plants prokaryotes, SOD2 and Nha1 in fungi, and AtNHX1 and SOS1 in Ara- as a source of genes for crop improvement for enhanced salt tolerance. bidopsis (Wu et al., 2005). However, engineering plants for over- But in fact, transferring salt-tolerant genes from wild relatives to expression of genes for different types of antiporters has recently domesticated crops is not so easy because of reproductive barriers gained a ground as an effective approach for controlling the uptake of (Ashraf et al., 2008) and thus very few examples could be tracked down toxic ions and hence enhanced plant salt tolerance. In Table 1, from the literature (Ashraf, 1994; Flowers, 2004) wherein this approach transgenic lines of different crops with engineered genes for different had been effectively utilized. Chinnusamy et al. (2005) have listed some types of transporters are listed. Some scientists have shown high conclusive reasons for limited success in improvement of crop salt resistance of engineered lines to salt stress, but they have not tolerance through conventional breeding. The approach as a whole is measured growth performance of the lines in terms of biomass time-consuming and labor-intensive; undesirable genes are often production or seed yield (in case of grain crops) under saline transferred in combination with desirable ones; and reproductive conditions. In fact, they measured only germinability or survival of barriers limit transfer of favorable alleles from inter-specificandinter- seedlings in saline regime under controlled conditions. For example, generic sources. Due to these reasons genetic engineering, as an Bao-Yan et al. (2008) and Gu et al. (2008) have generated two alternative strategy to conventional breeding, is being employed transgenic lines of Arabidopsis using MsNHX1 from alfalfa (M. sativa) emphatically worldwide these days not only for improving stress and ZmOPR1 from (Zea mays), respectively. They have shown tolerance but also for improving quality and yield potential of most crops. considerable improvement in seed germination of both transgenic lines under saline conditions. Both studies were in fact conducted 3. Engineering plants for enhanced salt tolerance: under controlled growth room conditions, and the plant performance transgenic approach was not appraised as adult or under natural field conditions. Furthermore, other scientists have shown a substantial improvement Transgenic plants are those plants that have been genetically in growth and biomass production in engineered lines of different altered by inserting desired genes directly into a plant cell. Such plants crops under saline regimes. For example, Verma et al. (2007) have 746 M. Ashraf, N.A. Akram / Biotechnology Advances 27 (2009) 744–752

Table 1 Improving plant salt tolerance through engineering genes for ion transporters.

Gene engineered Transgenic Source Trait improved Growth Growth improvement in Reference host conditions for transgenic lines under testing salt stressa Vacuolar Na+/H+ Arabidopsis Alfalfa (Medicago Increased osmotic adjustment and MDA Laboratory Germination and seedling Bao-Yan antiporter MsNHX1 thaliana L. sativa L.) content conditions growth et al. (2008) Vacuolar Na+/H+ Tall fescue A. thaliana L. Higher root sodium contents and activity Hydroponics About 18% improvement in Zhao et al. antiporter AtNHX1 (Festuca of vacuolar Na+/H+ antiporter in greenhouse shoot dry weight, and 43% in (2007) arundinacea root dry weight Schreb.) Vacuolar Na+/H+ ( Atriplex gmelini Eight-fold higher activity of the vacuolar-type Greenhouse 50% or 81–100% survival of Ohta et al. antiporter AgNHX1 sativa L.) Na+/H+ antiporter and seedlings (2002) hydroponics in growth room Vacuolar Na+/H+ Rice (O. Pennisetum glaucum Extensive root system Hydroponics About 81% improvement in Verma antiporter PgNHX1 sativa L.) (L.) R. Br. in greenhouse shoot and root lengths et al. (2007) Vacuolar Na+/H+ Wheat A. thaliana L. Improved germination rate, biomass production, Greenhouse About 68% increase in shoot Xue et al. antiporter AtNHX1 (Triticum grain yield, and leaf K+ accumulation, and and field dry weight and 26% in root dry (2004) aestivum L.) reduced leaf Na+ conditions weight OPR1 cDNA A. thaliana L. Maize (Zea mays L.), Improved seed germination and altered Hydroponics Improved germination Gu et al. designated as inbred line, Han 21 transcription of RD22, RD29B and ABF2 in growth (2008) ZmOPR1 room Vacuolar Na+/H+ Tall fescue A. thaliana L. Better growth at 200 mM NaCl Growth room Transgenic plants thrived well Tian et al. antiporter gene (F. and at 200 mM NaCl (2006) AtNHX1 arundinacea greenhouse Schreb.) EhCaBP for calcium Tobacco Entamoeba histolytica Enhanced rate of germination, and growth. Growth room About 20–37% increase in dry Pandey binding protein (Nicotiana weight et al. (2002) tabacum L.) Vacuolar Na+/H+ Perennial Rice (O. sativa L.) Higher concentration of Na+,K+ and proline Growth room Transgenic plants survived at Wu et al. antiporter gene ryegrass 350 mM NaCl but all wild type (2005) OsNHX1 (Lolium plants died perenne L.) Vacuolar Na+/H+ Tobacco (N. Cotton (Gossypium Na+ vacuolar sequestration Growth room About 100% increase in dry Wu et al. antiporter GhNHX1 tabacum L.) hirsutum L.) weight/plant (2004) Vacuolar Na+/H+ Tomato A. thaliana L. Overproduction of vacuolar Na+/H+ antiport Hydroponics Increased growth, and flower Zhang and antiporter AtNHX1 (Lycopersicon protein in greenhouse and seed production Blumwald esculentum (2001) L.) Vacuolar Na+/H+ Tobacco (N. Aeluropus littoralis Compartmentalized more Na+ in the roots and Greenhouse About 150% increase in relative Zhang antiporter AlNHXI tabacum L.) kept a relative high K+/Na+ ratio in the leaves dry weight/plant et al. (2008) Vacuolar H+- Alfalfa (M. A. thaliana L. High Na+,K+ and Ca2+ in leaves and roots Growth room About 215% improvement in Bao et al. pyrophosphatase sativa L.) shoot dry weight (2009) AVP1 Vacuolar Na+/H+ Rice (O. Wild type rice (O. Growth in transgenic cells or plants and Hydroponics Maintained growth at 0.2 M Fukuda antiporter OsNHX1 sativa L.) sativa L.) accumulated higher Na+ and lower K+ in growth NaCl while wild type plants et al. (2004) room died Yeast A. thaliana L. Yeast Better in fresh and dry biomass, and accumulated Greenhouse Transgenic plants were 87% Gao et al. (Schizosaccharomyces (Schizosaccharomyces less Na+ and more K+ higher in plant fresh weight (2003) pombe) sodium 2 S. pombe) and 27.5% in dry weight SOD2 Plasma membrane Na+/ A. thaliana L. Wild type (A. Improved germination rate, root growth, Greenhouse About 11–15.4% increase in Shi et al. H+ antiporter SOS1 thaliana L.) protein level, chlorophyll contents and root growth (2003) plant survival rate, reduced accumulation of shoot Na+ Vacuolar Na+/H+ Brassica A. thaliana L. Improved plant growth, seed yield and Greenhouse About 2.3% higher in plant Zhang antiporter AtNHX1 napus seed oil quality; toxic effect of Na+ was fresh weight, and 2.34% in et al. (2001) mitigated and increased proline grain yield at 10 mM NaCl accumulation, and lower cytosolic K+ Vacuolar Na+/H+ Maize (Z. A. thaliana L. Germination percentage Field At 0.5% NaCl, germination Xiao-Yan et antiporter AtNHX1 mays L.) capacity of transgenic plants al. (2004) was about 80% while that of wild plants was only 13–57% Vacuolar Na+/H+ Tobacco (N. B. napus L. Improvement in growth, number of flowers Greenhouse Transgenic plants grew better Wang antiporter BnNHX1 tabacum L.) and yield and produced seeds at et al. (2004) 200 mM NaCl, while wild type plants died. Vacuolar Na+/H+ Tobacco (N. Hordeum Improved growth, leaf Na+ and K+ contents, Greenhouse Dry weight of transgenic Lu et al. antiporter cDNA gene, tabacum L.) brevisubulatum Na+/K+ ratio and proline contents seedlings increased by 1.8-fold (2005) HbNHX1 (Trin.) Link Vacuolar H+- A. thaliana L. A. thaliana L. Higher levels of Na+,K+ and Ca2+ in leaves, Growth room Transgenic plants grew well at Gaxiola pyrophosphatase and relative water contents; protein contents 250 mM NaCl, while wild et al. (2001) AVP1 increased about 1.6–2.4-fold, While osmotic plants died after 10 days of salt potential and stomatal aperture decreased treatment

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Table 1 (continued) Gene engineered Transgenic Source Trait improved Growth Growth improvement in Reference host conditions for transgenic lines under testing salt stressa Plasma membrane Na+/ Rice (O. Yeast Accumulated higher K+ (29.4%), Ca2+ (22.2%), Greenhouse 32.5% increase in shoot fresh Zhao et al. H+ antiporter sodium sativa L.) (S. pombe) Mg2+ (53.8%) and lower Na+ (26.6%) contents weight at 150 mM and 57.1% at (2006) 2(SOD2) and enhanced net photosynthetic rate (100%) 300 mM NaCl and decreased MDA level (57.10%)

This table is partly expanded from Yamaguchi and Blumwald, 2005 with permission. a Calculated from the original data presented in each study. shown about 81% improvement in shoot and root lengths in transgenic organic solute or they are unable to produce it, engineering for rice line containing PgNHX1 from Pennisetum glaucum, under salt overproduction of such organic compound seems to be a plausible stress. Similarly, two tobacco (Nicotiana tabacum) transgenic lines approach. In the last few decades, there has been a growing interest in (GhNHX1, Wu et al., 2004; AlNHX, Zhang et al., 2008) showed about the metabolism of organic solutes synthesis as a means of engineering 100 and 150% higher plant dry weight than the wild type when tested salt tolerance in crops. In Table 2, a number of transgenic lines of at 300 mM and 400 mM NaCl, respectively. A marked improvement different crops are listed which show overproduction of a specific (about 215%) has been recently observed in shoot dry weight of organic solute and hence enhanced salt tolerance. Of various compatible transgenic line of alfalfa overexpressing vacuolar H+-pyrophosphatase organic solutes, proline, glycinebetaine and trehalose have been most gene, AVP1 from Arabidopsis when tested under saline conditions (Bao favored for transformation experiments, because of the fact that et al., 2009). All these studies and many other listed in Table 1 show accumulation of these solutes has been correlated well with tolerance remarkable performance of transgenic lines of different crops under to salt stress and other stresses in plants. For example, proline has been saline regimes, but as is evident from the information depicted in widely reported as one of the potential indicators of salt tolerance in a Table 1 almost all studies were carried out under growth room or number of plant species (Ashraf and Foolad, 2007). Therefore, over- greenhouse conditions and the performance of the transgenic lines production of proline in plants may lead to enhanced salt tolerance in was not tested under natural field conditions. However, there is only plants. With this objective in mind, many biologists have overexpressed one study by Xue et al. (2004) in which the performance of a wheat (T. Δ1-pyrroline-5-carboxylate synthetase (P5CS), a dual function enzyme, aestivum) transgenic line overexpressing vacuolar Na+/H+ antiporter which catalyzes the conversion of glutamate to Δ1-pyrroline-5- gene, AtNHX1 from Arabidopsis, was appraised under both greenhouse carboxylate, which is finally reduced to proline. For example, the gene and natural field conditions (Table 1). for P5CS has been overexpressed in rice (Su and Wu, 2004), potato It is evident that the degree of salt tolerance varies at different (Hmida-Sayari et al., 2005), wheat (Sawahel and Hassan, 2002), and phases of life cycle of most plant species (Ashraf et al., 2008), so it is tobacco (Kishor et al., 1995; Hong et al., 2000). Indeed, all transgenic not certain whether most of the transgenic lines that have shown lines accumulated proline many fold higher than that in wild type plants remarkable performance under saline conditions at the initial growth and showed a marked increase in growth under saline conditions. Genes stages (germination and seedling growth) would also maintain their encoding key enzymes of glycinebetaine biosynthesis such as choline degree of salt tolerance when tested as adult. Also it is not sure dehydrogenase, choline oxidase, and choline monooxygenase have been whether the transgenic lines tested under controlled growth room or engineered (Hayashi et al., 1997; Nuccio et al., 1998; Bhattacharya et al., greenhouse conditions would perform similarly under natural field 2004). Plants such as arabidopsis (Huang et al., 2000), cabbage conditions as the latter are exposed to a multitude of environmental (Bhattacharya et al., 2004), rice (Sakamoto et al., 1998), and Brassica factors other than the salt stress. juncea (Prasad et al., 2000) so transformed showed enhanced salt tolerance. Trehalose, a nonreducing disaccharide, has recently gained a 5. Transgenic plants for compatible organic solutes ground as a potential osmoprotectant analogous to proline and glycinebetaine, because its overproduction in plants has been shown Of a number of plant responses to salt stress, overproduction of correlated with salt tolerance. For example, a transgenic rice line different types of compatible organic solutes is the most common one expressing the trehalose gene exhibited enhanced tolerance not only to (Ashraf and Foolad, 2007). The most common organic solutes that play salinity stress, but also to drought and cold stress (Garg et al., 2002). an active role in the mechanism of plant salt tolerance include proline, Similarly, another transgenic rice line expressing chimeric gene Ubi1:: trehalose, sucrose, polyols and quaternary ammonium compounds such TPSP thereby showing high accumulation of trehalose exhibited as glycinebetaine, prolinebetaine, alaninebetaine, hydroxyprolinebe- enhanced co-tolerance to drought, salt, and cold (Jang et al., 2003). taine, pipecolatebetaine, and choline O-sulfate (Rhodes and Hanson, Similarly, different plants have also been transformed for overproduc- 1993). Although increased accumulation of such organic solutes in tion of mannitol, myo-inositol, sorbitol etc. All these engineered plants plants under salt stress is widely reported, some plant species synthesize also exhibited increased salt tolerance. and accumulate very low quantity of these compounds, while some It is worth noting from the transgenic lines listed in Table 2 that others naturally do not synthesize them under non-stress or stress most of transformation experiments have been carried out with the conditions (Rhodes and Hanson, 1993; Ashraf and Foolad, 2007). For well known model species such as arabidopsis and tobacco. Only very example, glycinebetaine, one of the most common quaternary ammo- few food crops such as wheat and rice, have been focused for nium compounds, is known to naturally accumulate in response to salt transformation experiments. Furthermore, all studies reported therein stress in many plant species, including sugar beet (Beta vulgaris), spinach were carried out under controlled conditions and none of the lines (Spinacea oleracea), (Hordeum vulgare), (Sorghum showing extraordinary performance in terms of overproduction of a bicolor), and wheat (T. aestivum)(Weimberg et al., 1984; Fallon and specific osmoprotectant and growth under controlled conditions has Phillips, 1989; Rhodes and Hanson, 1993; Yang et al., 2003; Ashraf and been tested under natural field conditions. Foolad, 2007). However, in contrast, some plant species such as rice (Oryza sativa), mustard (Brassica spp.), tobacco (N. tabacum), and 6. Transgenic plants for enhanced antioxidant production arabidopsis (Arabidopsis thaliana) are not capable of synthesizing GB under optimal or stress conditions (Rhodes and Hanson, 1993; Ashraf Despite affecting a multitude of physiological and biochemical and Foolad, 2007). Whether the plants produce low amount of an processes in plants, salt stress also triggers a number of degenerative 748 M. Ashraf, N.A. Akram / Biotechnology Advances 27 (2009) 744–752

Table 2 Improving plant salt tolerance through engineering genes for osmoprotectants.

Gene engineered Transgenic host Source organism Trait improved Growth Growth improvement in Reference conditions for transgenic lines under testing salt stressa A novel DREB (dehydration Tobacco Brassica juncea L. Transgenic plants accumulated Controlled Biomass or yield was not Cong et al. responsive element binding (Nicotiana 3.2–4.4-fold higher proline under conditions measured (2008) protein) gene, designated as tabacum L. cv. normal conditions and 2.5–2.9-fold BjDREB1B NC89) at 150 mM NaCl, and 46.6% higher chlorophyll under normal conditions while, 171.4% at 600 mM NaCl as compared to those in wild types Δ1-Pyrroline-5-carboxylate Arabidopsis Triticum aestivum L. Transgenic seedlings produced 3- Growth room About 25.5% improvement in Ma et al. reductase (TaP5CR) thaliana L.) fold more proline under normal relative root length at 50 mM (2008) conditions, while 2.5–4times NaCl, while 31.1% at 100 mM higher at 150 mM NaCl than those NaCl in wild types. In contrast, MDA contents decreased 16.6% more under normal, while 24% under saline conditions as compared to those in wild types Δ1-pyrroline-5-carboxylate Rice (Oryza sativa Moth bean (Vigna Transgenic plants accumulated Greenhouse About 30–93% improvement in Su and Wu synthetase (p5cs) L.) cv. Kenfong aconitifolia) higher concentration of p5cs and growth shoot fresh weight and 37–74% in (2004) mRNA and showed faster growth room root fresh weight at 200 mM of transgenic plants. About 120– NaCl 315% more proline content in transgenic plants under control and 163–216% at 300 mM NaCl as compared to that in wild type Mannitol-1-phosphate Loblolly pine Agrobacterium Improved survival rate, synthesis Greenhouse Plant survival rate of transgenic Tang et al. dehydrogenase (Mt1D) and (Pinus taeda L.) tumefaciens strain and accumulation of mannitol plants was about 78.1% at 85 mM (2005) glucitol-6-phosphate LBA4404 and glucitol, and enhanced and 62.8% at 120 mM NaCl after dehydrogenase (GutD) ability to tolerate high salinity in 15 weeks of salt treatment, while transgenic plants wild plants did not survive even after 9 week growth NADP-dependent sorbitol-6- Japanese Apple (Malus x Higher sorbitol contents (14.5– Growth room Improved growth was observed Gao et al. phosphate dehydrogenase persimmon domestica Borkh.) 61.5 µmol g− 1 f wt) at 50 mM at 50 mM NaCl (2001) (S6PDH) (Diospyros kaki NaCl in transgenic plants while Thunb.) absent in wild type. Fv/Fm was improved in transgenic plants Trehalose-6-phosphate synthase Tomato Yeast Higher trehalose contents Greenhouse Improved seed dry weight Cortina and (TPS1) (Lycopersicon (Saccharomyces (0.15 mg/g), improved plant (about 23.2%) Culianez- esculentum L.) cv. cerevisiae) growth and higher chlorophyll Macia UC82B (25–35%) and starch (35–65%) (2005) contents in transgenic plants at 100 mM NaCl as compared to wild types Δ1-pyrroline-5-carboxylate Potato (Solanum A. thaliana L. At 100 mM NaCl transgenic plants Greenhouse Improved plant growth. Hmida- synthetase (P5CS) tuberosum L. cv. showed 3–10 times increase in Reduction in tuber yield was only Sayari et al. Nicola) proline level as compared to that in 29–42% in transgenic plants, (2005) wild type plants. Transgenic plants while in wild type it was 63% also showed an improved under saline conditions tolerance to salinity through a much less altered tuber yield and weight compared to non- transgenic ones Choline dehydrogenase (betA) Cabbage (Brassica Escherichia coli Improvement in plant biomass, Greenhouse Improved plant biomass about Bhattacharya oleracea var. chlorophyll contents, and 21.3% at 150 mM and 20% at et al. (2004) capitata) cultivar relative water contents, and less 300 mM NaCl ‘Golden Acre’ negative water potential and osmotic potential of transgenic plants as compared to those of wild type plants at 150 and 300 mM NaCl Δ1-pyrroline-5-carboxylate Wheat (T. Moth bean (V. Transgenic plants expressed Greenhouse Transgenic plants were Sawahel and synthetase (P5CS) aestivum L.) aconitifolia) higher level of P5CS mRNA, and unaffected up to 200 mM NaCl Hassan P5CS protein, and accumulated and a small growth reduction (2002) 2.5-fold higher proline than that was observed at 250 mM NaCl, in wild type plants while wild type plants died or showed stunded growth at 100 mM NaCl bet gene cluster i.e., betA Fresh water E. coli Transgenic plants showed Culture Cell number increased 125.5% at Nomura (choline dehydrogenase), betB cyanobacterium improved cell growth even at medium 0.4 M NaCl et al. (1995) (betaine aldehyde (Synechococcus sp. 0.4 M NaCl and exhibited higher dehydrogenase), betI (putative PCC7942) activities of PSI and PSII at 200 mM regulatory protein), and betT NaCl than those in wild type plants (choline transport system) M. Ashraf, N.A. Akram / Biotechnology Advances 27 (2009) 744–752 749

Table 2 (continued) Gene engineered Transgenic host Source organism Trait improved Growth Growth improvement in Reference conditions for transgenic lines under testing salt stressa Mannitol 1-phosphate A. thaliana L. E. coli Mannitol (0.05–12.00 µmol g− 1 Growth room Transgenic seeds containing Thomas et al. dehydrogenase (mtID) fresh weight) was synthesized in mannitol germinated up to (1995) transgenic plants but not in wild 400 mM NaCl, while in wild type plants. Germination plants germination ceased at percentage, fructose, inositol, 100 mM NaCl and proline contents of transgenic plants were improved under 200 mM NaCl Δ1-pyrroline-5-carboxylate Tobacco (N. Moth bean (V. Transgenic plants synthesized Greenhouse Transgenic plants had 30% higher Kishor et al. synthetase (P5CS) tabacum L.) aconitifolia) 10–18-fold more proline and and growth root length,170% root dry weight, (1995) showed improved tolerance with room 254% capsule number and 42.1% respect to root length, root dry seed number per capsule at weight, capsule number and 500 mM as compared to those of number of seeds per capsule at wild type 500 mM NaCl as compared to wild type L-myo-inositol 1-phosphate Tobacco (N. Halophytic rice Transgenic plants had improved Greenhouse Growth improvement was Majee et al. synthase from P. coarctata tabacum L.) (Porteresia coarctata growth, about 2–7-fold increase and tissue observed in transgenic plants at (2004) (Roxb.) designated as PcINO1 (Roxb.) in the inositol contents and culture room 300 mM NaCl, but biomass was about 2-fold higher not recorded photosynthetic competence at 300 mM NaCl as compared to the non-transformed plants Choline oxidase (COX) A. thaliana L., (N. Arthrobacter pascens Improvement in growth, Growth 37.5% increase in shoot dry Huang et al. tabacum L., and photosynthetic rate and chamber weight of transgenic B. napus (2000) Brassica napus L. glycinebetaine synthesis in plants at 300 mM, 90.6% in A. transgenic plants thaliana at 100 mM and 200% in tobacco at 150 mM NaCl Δ1-pyrroline-5-carboxylate Tobacco (N. Moth bean (V. Proline content was 2-fold Tissue culture Improved germination rate Hong et al. synthetase (P5CS129A) tabacum L.) aconitifolia) higher than that in wild type. and growth (675%) and seedling fresh weight (2000) Transgenic plants also had room (333.3%) at 200 mM NaCl improved seedling growth, and enhanced malondialdehyde production at 200 mM NaCl L-2,4-diaminobutyric acid Tobacco (N. Halomonas elongata Improved accumulation of Tissue culture Transgenic plants had normal Nakayama acetyltransferase (ectA), tabacum L.) ectoine, increased tolerance to room growth pattern at 500 mM NaCl et al. (2000) L-2,4-diaminobutyric acid hyperosmotic stress, and better while in wild type plants it was transaminase (ectB) and growth in transgenic plants inhibited L-ectoine synthase (ectC) Myo-inositol O- Tobacco (N. Mesembryanthemum Improvement in methylated Hydroponics in Improved growth at 250 mM Sheveleva methyltransferase (ImtI) tabacum L.) crystallinum inositol D-ononitol content, greenhouse NaCl but data was not presented et al. (1997)

photosynthetic CO2 fixation and stable solute accumulation in transgenic plants at 250 mM NaCl

a Calculated from the original data presented in each study. reactions mediated by reactive oxygen species (ROS) such as super- tocopherol, and carotenoids, whereas antioxidant enzymes include − . oxide anion (O2 ), hydrogen peroxide (H2O2), hydroxyl radical ( OH), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and 1 and singlet oxygen ( O2). Although ROS in plants are generated under enzymes of ascorbate–glutathione cycle (Ashraf, 2009). Most of the optimal growth conditions and their concentration is usually found antioxidant enzymes can substantially lower the levels of superoxide low (Polle, 2001), most environmental adversaries trigger enhanced and hydrogen peroxide in plants (Ali and Alqurainy, 2006). production of ROS (Karpinski et al., 2003; Laloi et al., 2004). When a A number of studies that appeared in the literature during the last plant experiences a stress, ROS are produced through pathways such few decades have revealed that improvement in the salt tolerance of as photorespiration, mitochondrial respiration, and from the photo- different plant species is possible through engineering the genes for synthetic apparatus (Ashraf, 2009). One of the most prominent effects specific enzymes which can effectively scavenge reactive oxygen of salt stress on plants is that it limits the supply of CO2 to the leaf by species (Van Camp et al., 1994; Foyer et al., 1994, 1995; Polidoros and closing the stomata, which in turn causes overreduction of photo- Scandalios, 1999). However, some studies listed in Ashraf (2009) synthetic electron transport chain, thereby resulting in the generation provide sound evidence that overexpression of genes for antioxidant of reactive oxygen species (Halliwell and Gutteridge, 1985; Thompson enzymes causes enhanced salt tolerance in different crops. For et al., 1987). These ROS are cytotoxic and can adversely affect normal example, dehydroascorbate reductase (DHAR), an enzyme that metabolism by causing a substantial oxidative damage to biomole- reduces the oxidized ascorbate in plant cells, is vital for recycling cules such as membrane lipids, proteins, and nucleic acids (McKersie ascorbate. In a recent study, Ushimaru et al. (2006) have shown that and Leshem, 1994). Despite being toxic, ROS play a vital role in various the expression of rice DHAR in transgenic arabidopsis enhanced the important physiological phenomena such as cell signaling, gene degree of salt tolerance measured as germinability under salt stress. regulation, senescence, apoptosis, pathogen defense, etc. (Dat et al., While engineering genes for different types of superoxide dismutase 2000; 2003; Laloi et al., 2004; Gechev et al., 2006). However, plants in plants such as rice (Tanaka et al., 1999), tobacco (Badawi et al. have their innate systems for scavenging/detoxifying the ROS, which (2004), Chinese cabbage (Tseng et al., 2007), and arabidopsis (Wang are commonly referred to as antioxidants (Asada, 1999). The most et al., 2004) a marked improvement in salt tolerance of these crops common antioxidants found in plants are ascorbate, glutathione, α- was achieved. However, in these studies salt tolerance was appraised 750 M. Ashraf, N.A. Akram / Biotechnology Advances 27 (2009) 744–752 either by the activities of specific enzymes involved in oxidative stress literature reflecting the testing of the performance of transgenic tolerance or just measured germination or seedling growth. Similarly, cultivars under natural field conditions. transgenic plants overexpressing glyoxalase pathway enzymes that The main reason for the limited success of producing salt-tolerant disallow an increase in accumulation of methylglyoxal (MG) have cultivars through genetic engineering is that in most cases only a been developed in different plants such as tobacco (Singla-Pareek single gene has been transformed, although it is now widely known et al., 2003; Yadav et al., 2005), Japonica and indica rice (Verma et al., that salt tolerance trait is multigenic in nature and a multitude of 2005), and Escherichia coli (Yadav et al., 2007). All transgenic plants/ physiological, biochemical and molecular processes are involved in organisms showed enhanced tolerance to salt stress, although in most the mechanism of salt tolerance (Zhu, 2002). Thus, the results of a cases early stage growth performance was measured. Only Singla- number of studies wherein improvement in salt tolerance has been Pareek et al. (2003) grew the transgenic tobacco plants till maturity shown many fold in terms of plant survival, germinability, biomass and recorded seed yield, but this study, like the other studies, was production or accumulation of a specific metabolite under saline carried out under controlled conditions. Genes for glutathione S- stress, should be used with some caution, unless the lines so produced transferase (Roxas et al., 2000) and catalase (Al-Taweel et al., 2007) are tested under natural stressful environments. It is highly likely that were also engineered in tobacco. The transgenic lines excelled the the performance of controlled conditions (laboratory or glasshouse) wild types in salt tolerance (Ashraf, 2009). tested transformed lines may differ under natural stress environments Although substantial increases in the activities of different antiox- due to the fact that the interplay of a number of edaphic and climatic idant enzymes in response to salt stress have been reported, some factors may influence overall plant growth and development (Ashraf controversies have been reported while drawing correlations of salt et al., 2008). tolerance with antioxidant enzyme levels (Gosset et al.,1994; Van Camp Furthermore, in majority of the studies, salt tolerance of transgenic et al., 1996; Fadzilla et al., 1997). Recently, while assessing the lines has been appraised using only sodium chloride, although it is performance of transgenic arabidopsis plants expressing the rice now well established that most soils contain significant amounts of dehydroascorbate reductase gene Ushimaru et al. (2006) reported salts other than sodium chloride (Buckman and Brady, 1967). For that excessive enhancement of the DHAR activity in transgenic plants example, in Pakistan depending on the nature of a dominant salt in a resulted in decreased tolerance to stress, and that an appropriate soil, salinity is mainly of two types, i.e. chloride salinity and sulfate quantity of DHAR expression is necessary for improvement of stress salinity (Mushtaq and Rafiq, 1977). Thus, performance of a transgenic tolerance. Thus, it is necessary to draw parallels between the degree of line evaluated in sodium chloride may not show such performance salt tolerance and the levels of antioxidant enzymes in engineered plants when tested on a natural salt-affected soil containing a mixture of so as to ascertain whether or not there is a progressive relationship salts or a predominant salt other than NaCl. In fact, it is evident from a between the two variables. number of earlier studies that the response a crop or a crop cultivar to As is evident from Ashraf (2009) only a single gene manipulation chloride or sulfate varies to a great extent (Curtin et al., 1993; Rogers has been done in most cases, but as a result of this a marked et al., 1998; Inal, 2002). improvement in salt tolerance has been shown. There is a consensus In majority of the reports cited in Tables 1 and 2, salt tolerance has from some researchers that not only one antioxidant enzyme activity been assessed at the initial growth stages, i.e., germination and needs to be enhanced to counteract oxidative stress, but in fact more seedling growth. Whether such degree of salt tolerance observed at than one related enzymes need to be enhanced for better perfor- the early growth stages is maintained at the later growth stages mance. For example, overexpression of SOD is only effective when the depends on the type of a species, because it has been earlier reported other related enzymes such as APX or catalase or both are also over- that in most plant species degree of salt tolerance varies with plant expressed (Badawi et al., 2004). development (Foolad, 2004; Cuartero et al., 2006; Ashraf et al., 2008). It is not surprising to note here once again that the transgenic lines Under the situation when salt tolerance of a crop varies with growth of different plants so produced for enhanced activities of antioxidant stage, there is a need to appraise salt tolerance at each growth stage so enzymes were tested under controlled growth room or greenhouse as to assess overall salt tolerance of the crop. conditions. Not a single line has been exposed to natural field While critically looking at all the reports presented in Tables 1 and 2 conditions wherein a number of edaphic and climatic factors have and many other such in the literature, it is evident that most of interactive effects on plant growth. transformation experiments have been carried out with the model plants such as arabidopsis and tobacco, and there have been 7. Challenges and prospects astounding successes in terms of enhanced salt tolerance. There is now a dire need to introduce these salt tolerance genes into crop Although substantial efforts have been made during the last plants. However, it is not sure what will happen when the genes century to develop salt-tolerant lines/cultivars of various crops using transferred to the model plants are expressed in cultivated species conventional plant breeding methods, there has been limited success (Flowers, 2004). Thus, the results regarding the salt tolerance of in achieving the desired goal. The reasons for the limited success transgenic arabidopsis or tobacco plants cannot be predictive of crop through conventional breeding have been enumerated in a number of species. Under such situation the sensible approach is to apply already published reviews (Flowers, 2004; Yamaguchi and Blumwald, transformation technology directly to a particular crop of interest so 2005; Ashraf et al., 2008). With the advent of molecular biology as to achieve a meaningful outcome. techniques it was presumed that developing stress-tolerant cultivars Many of the reports cited in the present review or others in the would be convenient and relatively less time consuming. However, literature show that while breeding or engineering for enhanced salt while critically reviewing the transgenic lines of different crops tolerance the key objective of almost all plant breeders and produced so far for different physiological pathways (Tables 1 and 2), bioengineers has been to produce crop cultivars/lines which will it is amply clear that again there has been a limited success in produce high biomass or seed yield on salt-affected soils. Appraisal of producing salt-tolerant cultivars through genetic transformation. The performance of such salt-tolerant cultivars on normal soils (non- ‘hyperbolic claims’ (Flowers, 2004) made in the literature in saline) is usually ignored, although farmers always desire to choose a developing transgenic plants with increased stress tolerance are cultivar that produces high yield on both normal and salt-affected soils. based simply on the performance of transgenic lines produced and The vital issue to be addressed is whether the manipulation of a tested under controlled conditions of growth room or greenhouse, and single or of many genes is necessary to modify complex traits like salt one cannot find any conclusive reports except the one listed in Table 1 tolerance. Supposedly, if a single gene transformation can substantially (wheat transgenic line tested in a field trial by Xue et al., 2004) in the lead to enhanced salt tolerance, this suggests either that altering the M. Ashraf, N.A. Akram / Biotechnology Advances 27 (2009) 744–752 751 levels of some fundamental components has an ample effect on a Blumwald E. Sodium transport and salt tolerance in plants. Curr Opin Cell Biol 2000;12: 431–4. number of other processes or that salt tolerance is not as complex as it Bohnert HJ, Shen B. Transformation and compatible solutes. Sci Hortic 1999;78:237–60. is in the real sense (Flowers, 2004). Buckman HO, Brady NC. The Nature and Properties of Soils. New York: The MacMillan Another key issue is that many of the stress-related metabolic Company; 1967. Cherian S, Reddy MP, Ferreira RB. Transgenic plants with improved dehydration-stress phenomena are not well understood yet. This is indeed one of the tolerance: progress and future prospects. Biol Plant 2006;50:481–95. major impediments of achieving highly salt-tolerant plants. Thus, an Chinnusamy V, Jagendorf A, Zhu JK. 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